A preponderance of recent evidence suggests that conduction block by local anesthetics is due to the charged cation rather than the neutral base. However the hypothesis that the cation is the active form does not explain the local anesthetic activity of benzocaine, nor account for reports that procaine is more potent at high than at low pH. The proposed studies are designed to test the hypothesis that local anesthetics are active at more than one site, specifically that the uncharged base contributes to conduction block by an action on membrane lipids. Extracellular recordings, enchanced by computer averaging techniques, will be used to monitor conduction in single lobster walking leg axons and in garfish olfactory nerves, two preparations with particularly favorable characteristics for this study. Conduction block will be quantitatively related to lipid configuration by the application of electron paramagnetic resonance spectroscopy to both spin-labeled nerves and synthetic phospholipid bilayers. Blocking activity due to the base will be discriminated from cationic block by examining a number of different local anesthetics with varying proportions of base to cation, including benzocaine, lidocaine, procaine and a quaternary analogue of lidocaine and by altering pH to produce a corresponding change in the base-to-cation ratio for agents with pka's in the range between 7 and 9. Exposure to hyperbaric conditions will be used to dissect out the portion of the block due to an action at a pressure-sensitive site. If the proposed studies succeed in apportioning conduction block between base and cation, concepts of local anesthetic action will have to be broadened to include at least two membrane sites. Structure-activity relationships of these drugs will be made clearer, and the information will aid in the rational synthesis of better local anesthetics. The results of the proposed experiments will also provide new insight into the membrane molecular constraints on normal impulse conduction.